This project is designed to develop optimal regulated gene inactivation for high throughput gene function studies in the two related pathogenic protozoa Trypanosoma brucei and Leishmania major. RNAi works well in T. brucei but does not inactivate gene expression in many cases. In addition, many attempts in a number of labs have been unable to achieve RNAi-mediated gene inactivation in Leishmania. We propose to develop endogenous cellular RNase P-mediated gene inactivation as a tool for assessing gene function in these two species. The approach exploits the efficient RNA cleavage activity of RNase P, which normally functions in tRNA maturation, but cleavage will be directed to specific mRNAs by a complementary external guide sequence (EGS) RNA. The interaction of the EGS with the target mRNA results in a structure resembling a precursor tRNA and consequently specific cleavage of target mRNA by RNase P. The EGS will be integrated into the parasite genome along with a regulatable promoter in order to control its production and hence mRNA cleavage. We propose to: (1) develop and assess external guide sequence interference (EGSi) as a tool for gene silencing in T. brucei and (2) adapt this approach to L. major. This proposal is designed to develop an alternative and/or complementary means to inactivate gene expression in T. brucei and L. major in a regulated fashion and at the RNA level to bypass complications due to these organisms being diploid. This project is expected to establish the proof of principal of this approach and it is designed to complement and supplement the gene discovery that is rapidly emerging from the Genome Sequencing Projects on these organisms.